Supporting element and associated method of use in microbiological, serological, immunological, clinical-chemical and similar laboratory work

ABSTRACT

For microbiological and other laboratory work in which supporting elements carrying an active substance is brought into contact with a substrate there is provided a supporting element of tubular or other shape and adapted to be forced through the substrate to the bottom of the same and thereby wholly or partly to confine an area of the substrate. The element carries the active substance on the side facing the confined area and isolates it from the substrate on the opposite side. The supporting element may comprise magnetic material and be pulled to the bottom of the substrate by magnetic force.

United States Paten Saxholm [4 1 Feb. 12, 1974 OTHER PUBLICATIONSHartman, Miniaturized Microbiological Methods" (Supplement 1 to Advancesin Applied Microbiology), p. 135-136, (1968).

Lab- Te ki dvertisement, J. Bacteriology 88(3):

[76] Inventor: Rolf Saxholm, Aastadveien 2,

Billingstad, Norway Gavin, Applied MlClOblOl. 5(l),25 33 (1957) 2 Filed;May 1 1970 Primary Examiner-Alvin E. Tanenholtz Assistant Examiner-MaxD. Hensley PP N05 33,594 Attorney, Agent, or FirmWaters, Roditi,Schwartz &

Nissen [52] U.S. Cl. 195/1035 R, 195/127, 23/230 B,

195/139, 23/253 R [57] ABSTRACT [51] Int. Cl ClZk 1/00 For microbiologrcal and other laboratory work in [58] held of Search 195M035 'fgggwhich supporting elements carrying an active substance is brought intocontact with a substrate there is f C d provided a supporting element oftubular or other 5 Memes shape and adapted to be forced through thesubstrate UNITED STATES PATENTS to the bottom of the same and therebywholly or 3,474,004 10/1969 Fink 195/139 LE partly to confine an area ofthe substrate. The element 3,401,087 9/1968 Kuzel et a1 195/139 LEcarries the active substance on the side facing the con- 3,106,84510/1963 Dimmick 195/139 LE fined area and isolates it from the substrateon the op- 2,87l,l68 H1959 Salisbury 195/139 LE posite side Thesupporting element may comprise 2,787,581 4/1957 Scherr 195/139 LEmagnetic material and be pulled to the bottom f the substrate bymagnetic force.

16 Claims, 15 Drawing Figures I [l I16 I I I I I w J & M 5/7 PATENIEDFEB 1 21914 "r 1 .936

SHEET 1 OF 3 I I I I INVENTOR memznrw 3,791,930

SHEET 2 BF 3 INVENTOR ATTORNEY PATE-NTEUFEBIZIBM $791,930-

- SHEET 3 UF 3 ATTORNEY SUPPORTING ELEMENT AND ASSOCIATED METHOD OF USEIN MICROBIOLOGICAL, SEROLOGICAL, IMMUNOLOGICAL, CLINICAL-CHEMICAL ANDSIMILAR LABORATORY WORK BACKGROUND OF THE INVENTION This inventionrelates to a new technique permitting rationalization and automation ofmicrobiological, serological, immunological, clinical-chemical andsimilar laboratory work.

Before the invention is explained, it will be expedient to give somegeneral background information.

Bacteria are grown in substrates (media) which are either liquid (broth)or of more of less solid consistency. Growth in liquid substrates takesplace throughout the entire substrate, while in solid substrates thegrowth usually takes place on the surface.

The solid substrates are made from the liquid by adding agar (or someother substance, such as gelatine) in various concentrations, or throughthe coagulation of albumin. There are a number of different types ofliquid and solid substrates. Often they have a common basic composition,but otherwise the composition changes according to which substances arerequired for the growth of the bacteria in question.

Many, though not all, bacteria decompose and utilize matter in differentways. This can be registered by various chemical or physico-chemicalreactions. As an example may be mentioned the fermentation or splittingof various types of so-called sugar (hereinafter called sugar) bybacteria. This fermentation, considered for the whole series of sugartypes in question, is often characteristic of the individual type ofbacterium, and frequently differs from one bacterium to the other. Thiscircumstance is widely exploited as an aid in distinguishing bacteriafrom one another.

When a bacterium decomposes a sugar to acid products, the pH value inthe substrate decreases, as may be observed by a change in the color ofan added indicator. If, for example, a sugar is added to a solidsubstrate as a fermentation basis, and bromothymol blue is added as anindicator, the result of inoculation with bacteria that break downsugar, and with bacteria that do not, is as follows: The sugar-splittingbacteria will grow on the surface of the substrate and form yellowcolonies, while the non-splitting bacteria will form colonies of thesame blue color as that of the substrate before inoculation. Yellow andblue colonies may lie close together, side by side, on the surface ofthe substrate.

A corresponding liquid substrate will change its color to yellow ifsugar-splitting bacteria grow in it, and will remain blue if the growthis purely of non-splitting bacteria.

When, as an aid to classification, fermentation determinations arecarried out, liquid substrates are usually employed.

Since one of the objects of the invention is to rationalize fermentationtests without limiting its scope to this field a more detaileddescription will be given of how a fermentation test is carried outaccording to a known method.

A pure culture of the micro-organism to be classified by thefermentation test must be prepared. Samples from this pure culture areinoculated into broths which have already been poured into test tubes.These broths contain an indicator and various types of sugar. The

test tubes are closed with plugs or caps. The test tubes containing thebroths are placed in racks and incubated for growth and reaction. Theresult is read by inspecting each tube, and is noted by hand.

This method requires that the test tubes be placed in the racks by hand.Each tube of broth contains only one kind of sugar, but the number ofkinds of sugar, and thus of tubes, may often be comparatively high. Thismeans that handling the tubes and segregating them according to type ofsugar when they are to be arranged in rows in the racks, can be arelatively laborious process. Furthermore, there is room for confusionand error.

The inoculation process is also relatively laborious. The inoculation ofeach test tube containing the sugar broth involves removal of thestopper, the upper edge of the tube being sterilized by a flame toremove any possible microbial contamination, and the stopper beingreplaced.

The test tubes, which are often of glass, must, after having been read,go through a time-consuming and laborious process before they can beused anew. First they are put in an autoclave to kill the cultures. Thenthey are emptied, washed, and rinsed, whereupon they are fitted withstoppers. After this they are sterilized and are once again ready toreceive new charges of broths containing various types of sugar andindicator.

The preparation of the substrates to be put into the test tubes alsodemands time and labor. First, the broth must be prepared and then, toportions of this, a number of different types of sugar have to be added.These mixtures are then poured into test tubes and duly sterilized. Thetubes are marked to indicate the type of sugar therein. They are thenplaced in a refrigerator, ready for use.

The test tubes require a good deal of space on ac count of theirrelatively large size and number. This makes itself felt in thelaboratory work, during storing in the refrigerator and incubator,storing and transport from producer to use. In addition, the racksrequire storing and cleaning.

The fermentation test described above can also be carried out bypositioning prefabricated bodies carrying the various types of sugar ina known solid substrate in a dish or container. The indicator may beconveniently present in the sugar bodies, or, alternatively, it may, inknown manner, have been added to the substrate in advance.

Before the bodies are positioned, the substrate surface in the dish isevenly inoculated all over with a suspension or broth containing a pureculture of the micro-organism.

After the bodies have been introduced onto the surface of the substrate,the sugar and the indicator if this has been added to the bodies willdiffuse out into the substrate. This takes place relatively fast in thesubstrates normally used. As a result, a successful reaction will notoccur if the sugar is applied via a paper disc into which it has beenabsorbed, or via a tablet into which the sugar has been mixed. Thediffusion must be delayed or limited.

SUMMARY OF THE INVENTION According to the invention, the substratearound the applied body is limited or confined, so that the sugardiffusing out will remain inside a peripherally closed chamber andequalize itself there with an evenly distributed concentration. Thus,this final concentration is dependent upon the volume of substrateenclosed and upon the quantity of the sugar applied.

A more detailed description according to the invention, will be givenhereafter. The limitation is achieved by means of supporting elementswhich encompass or confine a space closed laterally on all sides. Thesupporting elements are pressed down into the solid or semi-solidsubstrate, preferably so that their lower edges reach the bottom of thedish, while their upper edges preferably protrude somewhat above thesurface of the substrate. In other words, the supporting element shouldpreferably be somewhat higher than the depth of the substrate, which isgenerally about 0.5 cm or less. h ame e @911?). rsxame eii -1.99m orsome other suitable size. The supporting elements may be open or closedat the top.

The supporting elements contain those types of sugar which are to takepart in the reactions. Suitable concentrations and quantities of thesugars, for example, absorbed in filter paper or mixed into a suitablemass, are placed, during manufacture, in an expedient manner in thesupporting elements. This can be achieved, for example, by means of alining containing the sugar types and fixed to the inside of the wallsof the supporting element. This lining may, if desired, rest on a lowerinner shelf or step just above the lower edge of the supporting element.Alternatively, the sugars can be placed in hollows in the walls of thesupporting element, in which case internal wall lamellae are perforatedso that diffusion can take place into the space actually enclosed by thesupporting element. The bottom of the wall cavity may convenientlyconsist of the solid lower edge portion of the supporting element.

In addition to the walls which surround the supporting element to carrythe types of sugar, internal installations in the supporting element mayalso be used for the same purpose. Thus, in the center of the supportingelement, there may be mounted a torpedo-, projectileor star-shaped part,or other similar parts of expedient shape. They can be fixed, forexample, by means of a supporting spider carried by the upper edges ofthe supporting element. Furthermore, the supporting element can havepartition wall sections of various shapes which fully or partlysubdivide the space which the supporting element actually surrounds.These partition walls, and in fact all internal installations, may, likethe surrounding walls, be of hollow construction with perforated walllamellae.

After a supporting element containing a sugar has been placed in thesubstrate, diffusion takes place in the part of the substrate enclosedby the walls of the supporting element. In the case of supportingelements with perforated internal wall lamellae, diffusion takes placethrough these holes.

The amount of sugar which in one or another of the methods mentioned isintroduced together with the supporting elements during the productionprocess, is so adjusted that after diffusion into the substrate borderedby the walls of the supporting element, the sugar will assume aconcentration suitable for the fermentation tests to be carried out.

When carrying out fermentation tests, good results will be achievedirrespective of the geometrical form of the cross-section of thesupporting element, but even though this is so, it may be of interest todiscuss the shape of the supporting element in relation to diffusionequilibrium. The shape of the supporting element and the positioning ofthe sugar affects the rapidity with which the final concentration of thesugar is established in the substrate in the supporting element. Acircular supporting element, with the sugar placed along the wall,surrounds a quantity of substrate which will be relatively slowlypenetrated compared to non-circular shapes due to the fact that a circlehas a relatively large area compared to its circumference. Placing thesugar in the middle of the round supporting element and/or alongpartition walls in addition to along the inner wall of the supportingelement entails a more rapid state of diffusion equilibrium. Also, forexample, a narrow rectangular supporting element, with the sugar placedalong the walls, will be favorable for a relatively rapid establishmentof the final sugar concentration in the substrate enclosed. With certainreactions other than fermentation tests, the choice of an expedientshape of supporting element may have practical consequences for thecourse and outcome of the reactions.

An actual or positive fermentation reaction is revealed by change ofindicator color after incubation at 37C for a suitable period, while noor negative reaction is characterised by no change of color. The resultsof reactions are easy to read.

In a container containing a substrate, for example, a Petri dish, anumber of supporting elements are placed at suitable mutual distances.Each supporting element contains one type of sugar, and thus all thesupporting elements together represent the different types of sugar withwhich the fermentation test with the bacterium culture in question is tobe made.

It is convenient to supply the supporting elements to the substrate froma dispenser similar to those normally used for positioning paper discscontaining antibiotics. The supporting elements containing the sugarsare stored under sterile conditions, stacked on top of one another inmagazines, each magazine containing supporting elements having the samekind of sugar. In the dispenser or depositing device there is room forseveral magazines side by side, each magazine containing a differenttype of sugar.

Especially if delicate substances are contained in the supportingelements, it may be preferable to store them not only under sterileconditions in magazines, but also vacuum-packed.

After the supporting elements have been positioned on the surface of thesolid substrate they may, by hand or machine, be pressed so that theypenetrate the substrate and establish contact with the bottom of thedish, with the help of, for example, a plunger or similar device whichpresses down all the supporting elements in one and the same operation.The upper edge of the supporting elements, according to the invention,should preferably protrude somewhat above the surface of the substrate,so there is usually no danger of the surface of a plunger contacting thesubstrate.

If required, the supporting elements may be provided, at their loweredges, with small anchoring feet in order to prevent rebound from thesurface of the substrate.

The supporting elements may be mounted in a supporting skeleton by meansof which they can be simultaneously pressed to penetrate the substrate.

In accordance with the invention in US. Pat. application Ser. No.705,539 now abandoned, the supporting elements may be made to respond tomagnetic forces or, in other words, they may be made magneticallyresponsive. By magnetic forces, they are attracted to penetrate thesubstrate and to make contact with the bottom of the dish. If required,the magnetic force may be applied after the supporting elements, bygravity, have landed on the surface of the substrate. The magnetizableparts can be placed, for example, inside the wall of the supportingelement at its lower part or edge, and around the entire circumference.Alternatively, for example, the entire wall of the supporting elementmay be ferro-magnetic. This material may conveniently have the form ofan unbroken iron ring or sleeve, or also of granular parts, the maximumsize being determined by the thickness of the tubular wall.

Below and along the upper, outer edge of the supporting elements, theymay be provided with a collar of iron of suitable size and shape inorder to increase the response of the supporting element to the magneticfield. If required, the collar may carry slim, downwardly projectingsupporting studs extending outside the element and terminating flushwith the bottom thereof. Their number may, for example, be four, andthey may expediently be placed opposite one another so as to offer abalanced support.

It may be convenient to make the lower edge of the supporting elementfairly sharp or bevelled so as to facilitate penetration of thesupporting element through the substrate.

The supporting elements are discarded after use. Except for the abovementioned magnetizable components, they can, with advantage, be made ofplastic or the like, which is destroyed by burning.

The sugar fermentation reactions are not critically dependent uponcomplete contact or sealing between the bottom of the dish and the loweredge of the supporting element.

Even a roughly cut lower edge of a supporting element ensures reactionsof just as good quality as those with very good contact. Nevertheless itis possible, if desired, to establish adhesion between the lower edgeand the bottom of the dish.

The identities of the reactions are determined automatically orsemi-automatically in accordance with the invention described in US.Pat. application Ser. No. 705,539 now abandoned by means of definedpositions.

Also the outcome of each reaction is a function which may be read bymachine in accordance with the said US. Pat. application Ser. No.705,539 now abandoned. The readings are made as indicated in the patentapplication, preferably under visual control. The data machine usedindicates in a simple manner whether a reaction is positive or negative,or the degree of positivity shown by the reactions. In such a case asthis, with clear, distinct color reactions and well defined criteria fordetermining the outcome of a reaction, it may, in accordance with theabove mentioned patent application, be possible to take the readingsfully automatically.

conventionally, when carrying out fermentation tests, one investigationis made for each type of sugar. A defined, convenient concentration ofthe sugar is used. Investigations carried out by the inventor indicatethat it would be possible to achieve a better differentiated diagnosticexpression if several concentrations of the particular sugar were usedin the test. Such a method would be prohibitive with the working methodsavailable by present day techniques. Even when only one concentration foeach kind of sugar is used, fermentation tests impose a heavy load onthe laboratories.

The invention, however, makes it possible in a simple and labor savingway to carry out a fermentation test with several concentrations of onekind of sugar. Apart from using a separate supporting element for eachsugar concentration, it is possible to use one supporting element forall concentrations, the supporting element being divided into separatechambers to carry the sugar concentrations. The supporting element mayin such a case, for example, have the shape of a relatively longrectangular structure divided into consecutive transverse rectangularsections. Each section contains a given concentration of the sugar insuch a way that there is successive increase or decrease in the sugarconcentration from section to section in the long rectangular supportingelement. This supporting element represents therefore a particular typeof sugar, and its sections, various concentrations of that sugar.

The type of sugar is identified by the position of the rectangularsupporting element as such, and the concentration of sugar where apossible fermentation first occurs is determined by the position of thechamber concerned in the section row.

The supporting element under discussion may also be used with differenttypes of sugar in the chambers.

However, as mentioned above, the invention is not restricted to use infermentation tests, but is applicable to a large number ofmicrobiological, serological, immunological, clinical-chemical andsimilar tests.

For example, the method could be applicable to sensitivitydeterminations of bacteria towards antibiotics (and chemotherapeutics).

Today such determinations are usually made by means of discs containingantibiotics. A mechanical and automatic embodiment of this method isdescribed in US. Pat. application Ser. No. 705,539 now abandoned. Theprinciple of the antibiotic disc method is to measure the diameter ofthe inhibition zone around the antibiotic disc and use this measurementas an expression of the sensitivity of the bacterium towards theantibiotic concerned, of which the disc contains a given amount.

In contrast to this, the present invention permits sensitivitydeterminations to be carried out in a simple manner by means of atitration technique. In this manner, a quantitative determination isachieved in terms of different concentrations of an antibiotic. In somecases this is more advantageous than the conventional method involvingmeasuring the size of the zone diameter. The methods supplement oneanother.

According to the invention, supporting elements containing variousconcentrations of any antibiotic may be manufactured in advance for usein connection with the quantitative technique discussed above. In atitration test a given series of supporting elements is used for eachantibiotic in such a way that the concentration of the antibiotic inquestion increases from one supporting element to the next. In additionto individual supporting elements, a supporting element subdivided intosection chambers, as described above, can also be used for this purpose.The chambers have been supplied in advance with various concentrationsof an antibiotic increasing in steps from chamber to chamber.Observations are made of the lowest concentration which inhibits growth,assuming that the bacterium being investigated is sensitive towards theantibiotic in question.

Today, if it is desired to carry out a quantitative sensitivityexamination of the kind mentioned, this must be done in a series of testtubes containing broth, or in containers holding solid substrate,containing concentrations of antibiotic increasing in steps. On accountof the labor this involves, this present-day technique would beprohibitive as a general routine method. Yet, in spite of the amount ofwork involved, it is used when determining the sensitivity of tuberclebacilli.

This is due to special circumstances to be described below. Theinvention, however, makes it possible, in a simple manner, to make useofa titration technique for determining the sensitivity of tuberclebacilli.

The reason why sensitivity determinations of tubercle bacilli towardsantibiotics differ from the sensitivity determinations of otherbacteria, is the relatively very slow growth rate of tubercle bacilli.One consequence of this fact is that it is not convenient to determinethe sensitivity of tubercle bacilli by the antibiotic disc method in theconventional manner.

However, the present invention makes it possible to determine thesensitivity of tubercle bacilli also by measuring the size of the zoneinhibited. This can be achieved by using a relatively long and narrowrectangular supporting element as a support for an antibiotic. Theantibiotic is fixed to the inside of one of the short sides of thesupporting element. The diffusion of the antibiotic concerned is guidedby the shape of the supporting element in a compressed volume along thelongitudinal axis of the rectangular supporting element. The length ofthe zone inhibited is a measure of the sensitivity. The supportingelements may be open or closed at the top, the closure, when present,being in the form of a flat transparent roof. This cover will delay thedesiccation of the substrate and reduce the risk of contamination duringthe incubation period of several weeks at 37C which is necessary forpermitting the growth of the tubercle bacilli to develop. In itself, thesealing of the substrate dish is not entirely sufficient to affordprotection against a certain degree of desiccation during such a longperiod of incubation. Additional sealing of the dishes is required (forexample with tape) or they can be incubated inside plastic bags orboxes. These measures can be rendered unnecessary by using supportingelements closed at the top as mentioned above.

This invention also lends itself to an expedient application of aserological technique, particularly an immuno-diffusion or precipitationtechnique. This very important technique has a multifarious andincreasing application, but it is laborious and difficult to use inseries tests. With the help of this invention, this technique will bemade available in a great many fields for routine laboratory work andwill, to a significant degree, contribute to increasing the breadth ofinformation concerning laboratory investigation and research and toenhance their significance.

Instead of inoculating the substrate (preferably at the surface) with abacterium culture, it is possible, in this case, to spread a quantity ofantigen or antibody over the surface of the substrate, or mix it in withthe substrate before pouring it into the dishes, all according towhether the supporting elements contain antibody or antigenrespectively. When employing a quantitative precipitation technique, theprefabricated supporting elements contain graded quantities of the saidsubstances.

It is only necessary for the substrate to be a nutrient when microbialgrowth is to take place. In addition to the compositions referred toearlier, it may be made on the basis of cellulose, starch or othersuitable substances. Generally speaking the composition shall consist ofa material which permits diffusion and gives it a solid or semi-solidconsistency. The latter is usually somewhat elastic, but it may also beplastic. Furthermore, the substrate must be penetrable by the supportingelements.

In some cases, it may be sufficient to use supporting elements that arepartially open laterally, for example, rectangular supporting elementsfrom which the one short wall is missing, or which form open curves.Supporting elements of such a shape will constitute something betweenthe fully open ones, known per se, for example, paper discs, and thosedescribed above which surround a space closed on all sides. The materialwhich is to diffuse is fixed to the far wall of the supporting element,and the diffusion, in its first phase, is guided in a fixed direction bythe walls of the supporting element. When the diffusion has extendedbeyond the scope of the supporting element, it will spread in alldirections.

The supporting elements may be of various shapes, each suited forspecific diffusion experiments. This may apply to differentimmunodiffusion tests but is not limited to this particular application.Thus, it may be very useful to consider and make use of the sameexpedient when bacterial growth is involved in the experiments, forexample, when testing synergism or antagonism between antibioticstowards bacteria. This investigation is an important supplement to theusual sensitivity determinations, and the possibility now available toinclude such tests in normal routine investigations is a large andimportant step forward.

The invention may also be used in connection with investigationsinvolving growth factors. In advance, growth-promoting substances can beplaced in the peripherally, for example fully, closed supportingelements. After placing the supporting elements in the inoculatedsubstrate there will, through diffusion of the substances mentioned intothe basic substrate in the dish, be produced new substrates in thevarious supporting elements, different from that originally present inthem. These new substrates consist of the basic substrate and one ofmore additional different components all dependent on the number ofgrowth-promoting substances placed in the supporting elements. Differentsubstrates are thus produced in one dish. Observations are then made ofthe inoculated bacterium culture in the different supporting elements tosee whether they react with abundant, unchanged or possibly reducedgrowth, in response to the addition of the various sub stances. Thetechnique may be used in connection with diagnosis or for purelyexperimental purposes.

Some test methods require a secondary application, to the reactionregion, of a particular substance (or substances) after a suitableperiod (or periods) in the course of the reaction, for example, afterincubation for growth has taken place. Such a technique may be carriedout by means of the invention. By using a dispenser, the secondaryapplication can be made in the same pattern as the one used for thefirst positioning. For example, paper discs or other suitable bodiescontaining the substance or substances to be added, may be placed in thesupporting elements. Another way of making secondary applications is touse a device having a number of inoculation points or pipettes arrangedin the same pattern as that in which the supporting elements wereplaced.

This technique, characterized by the secondary application of substancesafter suitable periods of time have elapsed, may also be useful in thecase of a quantitative method utilizing supporting elements whichcontain a substance present in a graded series of concentrations fromone supporting element to the next. The possibility is thereby opened ofadapting the method to different, relatively complicated titrationtechniques which today are carried out with liquid substances in testtubes. This also applies, for example, to reactions such as those inwhich red blood corpuscles are used to bring about haemolysis orhaemagglutination as an aid to determining the titration end-points.Secondarily, the red corpuscles may be placed on the surface of thesubstrate within the supporting elements, or they may have been admixedwith the substrate in advance. The substrate may have a very simplecomposition. It may be adapted to existing requirements. Theconcentration of, for example, the agar may be made to suit prevailingconditions. It may, for example, be relatively low. The substrate mayalso contain substances other than the red corpuscles mentioned above,which take part in the reactions in constant quantities from reaction toreaction.

Alternatively, titration techniques may be employed by using thesubstances secondarily applied in graded concentrations, while thesubstances contained in the supporting elements in this case form aseries with a constant concentration level.

Each supporting element need not contain only one substance, but maycontain several. These substances may be pooled, or positionedseparately at different and isolated places in the supporting element,in order to prevent a reaction from taking place between the substancesbefore diffusion into the substrate.

The supporting elements may also be produced without substances beingadded during the manufacturing process, but with the possibility ofadding required materials after the supporting elements have been placedin position in the substrate. With this object in view, the supportingelements may, for example, be supplied with a funnel-shaped inputchannel leading to a cavity in the wall of the supporting element where,in advance, a piece of absorbent paper may have been placed. Thesesupporting elements too, may be of various shapes, each of a designsuited for various specific diffusion experiments, for example, forexamining precipitation patterns, testing synergism or antagonismbetween antibiotics, or when examining growth factors. They may enclosea hollow space peripherally closed on all sides, or they may be partlyopen. The purpose is to give research workers a technical aid that canimprove possibilities and facilitate their work in the wide field ofexperiment covered by diffusion techniques and microbiology.

Furthermore, the method makes it easy to determine the level ofconcentration of antibiotics in tissue liquids and to carry out otherassays.

The method of the invention may be used in a quantitative and automatictechnique based on spectrophotometric readings through the contentswithin the posilltll tioned supporting elements. This may apply, forexample, to clinical-chemical tests. The system may be used for carryingout autoanalyzer work. Depending on how many supporting elements arepositioned by the dispenser at a time, the method may easily be used inconnection with a l2-channel run or more. The method may, for example,be used for screening tests.

The method of the invention is very flexible in use. It may be used bothwith substances which disperse rapidly, and with those which diffuseslowly in the substrate selected. This opens great possibilities formany different reactions and types of reaction to be adapted to thetechnique thus providing the inherent advantages which will be discussedin more detail below.

The method utilizes principles which were defined and explained in U.S.Pat. application Ser. No. 705 ,539 now abandoned. The advantagesdiscussed in the said application will also benefit the presentinvention. They are concerned with the automation of laboratory work andprimary noting of data taking into consideration a technicallydesirable, or even necessary human and visual guidance and control. Theyare further concerned with the possibility of establishing directcontact with an electronic data processing unit with enormous advantagesoffered thereby. Due to the present invention, these principles may finda more general and comprehensive application and will benefit a widerange of various microbiological, serological, immunological,clinical-chemical and similar tests and research in diffusion techniquesin biological and chemical fields.

A technique adapted to the invention will benefit by advantages inaddition to those mentioned above. These advantages relate partly to theactual laboratory work itself, partly to the preparation of thesubstrate, to cleaning, partly to questions of storage and transport andfinally to safety aspects in the laboratory work concerned, in that therisks of errors and confusion are greatly reduced.

A more detailed explanation of these conditions will be given withreference to the technique used in the bacterial fermentation of sugars,mentioned above. The invention involves only one inoculation for theentire series of sugars, as opposed to the system in use today, whichrequires one inoculation for each type of sugar, or, in some cases, fortwo or three types. Furthermore, there is only one cover, namely the lidof the Petri dish, to take off and replace, as compared with the presentpractice of using a plug or cap for each broth test tube correspondingto the individual sugar types inoculated. In addition, all thefermentation reactions are collected in one handy, convenient andspace-saving unit, the Petri dish, as compared with the present somewhatuntidy, laborious and cumbersome system with all the individual brothtest tubes to be handled and spread out one after the other in the holesof a rack. A further disadvantage in this connection is that theindividual test tubes containing the various types of sugar are alikeand can only be distinguished by means of a label indicating the type ofsugar contained. Relatively speaking, the number of substrate dishes tobe handled is small, and they are all of the same type, whereas thebroth containing test tubes are many, all externally the same, yetcontaining various types of sugar.

Although the reading and noting by the new technique may be carried outautomatically under visual controlor fully automatically according toU.S. Pat. application Ser. No. 705,539 now abandoned, these operationsare very laborious and time-consuming when the conventional method isused, in which the various fermentation tubes, standing in close rowsand columns in a rack arranged according to the kind of test and thetype of sugar, have to be removed manually one by one for takingreadings of the outcome and observing the identity, whereafter the dataare noted by hand.

With the conventional technique, the risk of error is relatively large.This applies to the reading and noting of data. It also applies to theinoculation. This is repeated for every broth test tube in the series.It is possible to overlook a tube, or it may be inoculated from a loopwhich does not contain sufficient inoculate to promote growth. Thisleads to erroneous results.

Errors and confusion may also occur at an earlier stage. They may occurwhen the technical assistant fetches the test tubes containing the sugarbroths. These are kept in a refrigerator and are grouped separately foreach type of sugar. It may however happen that the test tubes that havenot been used are put back in the refrigerator in a wrong group,inasmuch as the tubes in all groups have a similar outer appearance.Each test tube must therefore be checked.

Furthermore, errors and confusion may arise in the substrate departmentwhen the broths containing the various types of sugar are beingprepared. Mistakes may also be made in affixing labels and when thetubes are transported to the refrigerator.

The fact already mentioned that a relatively large number of brothscontaining different sugars are required for fermentation tests,increases the possibility of error. This applies particularly when thereare several specimens to be examined. In contrast to this, the techniqueaccording to the invention means that only a relatively small number ofPetri dishes have to be handled, and all of the same type.

The invention will result in a considerable saving in space both duringstorage in the laboratory and during internal and external transport anddispatch. The small supporting elements can be housed in magazines ascompact units as compared to the collection of relatively very largeindividual test tubes, which are awkward to handle. These require standswhich take up a lot of space and have to be kept clean and free fromcontamination. Often the tubes are of glass, and must, after use, be putin an autoclave, washed and sterilized before they can be used again.

On the other hand, it is very simple, after use, to remove and destroythe Petri dishes and with them the supporting elements placed in thesubstrate. When removing them, it is of considerable advantage to beable to handle the small units, requiring little space, which theinvention makes possible.

It is also very advantageous that the invention makes it possible to usea very simple and standardized substrate for various tests andexperiments.

Although other modifications or versions of the fermentation test exist,none of them or other laboratory methods currently in use or known haveany relation to the principles or method of the invention.

In the following, the invention will be described in more detail withreference to the drawings.

IN THE DRAWINGS FIG. 1 shows a Petri dish containing substrate, seenfrom above and in section along the line II in FIG. 2, and provided withsupporting elements according to the invention, for carrying out theexperiments desired.

FIG. 2 is a view in cross-section along line II--II in FIG. 1;

FIG. 3 is a vertical sectional view through one embodiment of asupporting element according to the invention;

FIG. 4 is a vertical sectional view through another embodiment of thesupporting element,

FIG. 5 is a vertical sectional view through another embodiment of thesupporting element;

FIG. 6 is a side elevational view of another embodiment of thesupporting element;

FIG. 7 is a top perspective view of another embodiment of the supportingelement;

FIG. 8 is a side elevational view, partly broken away and in section, ofanother embodiment of the supporting element,

FIG. 9 is a vertical sectional view through another embodiment of thesupporting element;

FIG. 10 is a vertical sectional view through another embodiment of thesupporting element;

FIG. 11 is a top perspective view of another embodiment of thesupporting element;

FIG. 12 is a section taken on line XII XII in FIG. 10;

FIG. 13 is a top plan view of another embodiment of the supportingelement;

FIG. 14 is a side elevation view of the embodiment of FIG. 13; and

FIG. 15 is a top perspective view of another embodiment of thesupporting element.

DESCRIPTION OF THE EMBODIMENTS The supporting element according to theinvention may be used for many tests and experiments, but in thefollowing description there is primarily given an account of thedetermination of bacteria on the basis of their fermenting effect onvarious types of sugar.

The different sugars are added to a solid or semi-solid substrate, whichis inoculated with the bacterium to be examined. The sugars are broughtinto contact with the substrate by means of a supporting elementaccording to the invention, for example, of the form shown in FIG. 3.The supporting element 3 in this embodiment is tubular and has on theinside of the wall thereof a lining 4 containing a sugar (and anindicator if this is not contained in the substrate) to be used in theexperiment. A ferro-magnetic ring 5 is mounted at the bottom of thetubular supporting element. By means of one or more permanent-magnets orelectro-magnets, it is possible to draw the tubular supporting elementsinto position through the substrate in a Petri dish. This positioning isshown in FIGS. 1 and 2. Here, the Petri dish is indicated by numeral 1.The substrate which has been inoculated with the bacterium culture to bedetermined, is indicated at 2. Six tubular supporting elements of thetype shown in FIG. 3 have been placed in the substrate. Each supportingelement 3 has a lining 4 carrying its own type of sugar. This variesfrom one supporting element to the next in the substrate dish. Theoutcome of the fermentation reaction (which sugars, if any, arefermenting) is a diagnostic aid when determining the bacterium.Fermentation is revealed by a change in the indicator color. Such achange has been indicated in the substrate which is encompassed orconfined by the supporting element designated at numeral 3' in FIG. 1.The fermentation causing the indicator to change color, is initiatedafter the various sugars and the indicator, if added to the bodies inthe linings 4 of the six supporting elements have diffused into thesubstrate enclosed by each individual supporting element, the diffusionof the sugar into the confined area producing an equilibriumconcentration thereof in the confined area.

The lining 4 may, for example, be of paper or cellulose, and it rests,in this embodiment, on a step or shelf formed at the lower end of thetubular supporting element 3 by the ring 5.

Instead of the lining, there may be provided a perforated wall lamellawhich forms the inner boundary of a cavity 4' in the wall of the tube 3aas shown in FIG.

4. The sugar is placedin the cavity. It is placed there.

either in its original form or it is absorbed in a lining or mixed withan inert mass. It diffuses through the perforated lamella and into thesubstrate encompassed by the tubular supporting element when this ispressed into the inoculated substrate.

The tubular supporting element may consist of various suitablematerials, for example, a plastic. The material may containferro-magnetic material, either inserted or cast in position, or thesupporting elements may be made of a ferro-magnetic material, such assteel.

If, in special cases, more powerful magnetic forces are required thanthose obtainable by the ferromagnetic ring 5 in FIG. 3, (even whenadding additional ferro-magnetic material into the wall of the tube) thetubular supporting element may, as indicated at 3b FIG. 5, have aferro-magnetic collar 12 or other suitably shaped member disposed aroundthe upper end of the tube. In FIG. 5 the entire wall of the tubularsupporting element, including the collar, consists of a ferro-magneticmaterial. The supporting element has, in this case, a somewhat taperedshape as compared to the one shown in FIG. 3. In FIG. 6 theferro-magnetic collar 12 is of triangular cross-section disposed on atubular supporting element 3c of the type shown, for example in FIG. 3.Should it be necessary to make the supporting element more stable whenit has a ferromagnetic collar at the upper end, the supporting elementmay be provided with four supporting legs 13 as shown in FIG. 7.

The embodiment in FIG. 8 has a projectile-like body It) placed centrallyin the tubular supporting element 3. This projectile-like body issupported from element 3 by a spider Ill. The body may be provided, onits outer face, with a coating containing the active, diffusiblesubstance corresponding to that in the lining 4. In this manner,diffusion equilibrium in the substrate mass is rapidly achieved.

Alternatively, different diffusible substances may be used in lining 4and on the surface of the body 10.

The body 10 may be hollow and perforated. The diffusible material maythen be placed in the body either during the manufacturing process or ata later stage. The supporting elements intended for the latter purposeare manufactured without active materials in the bodies 10. These meet ademand in certain situations, for example, in certain experiments wherethe experi menter wishes himself to add the materials to be tested. Thisis achieved by allowing them to drip down into the hollow bodies.

The supporting element as shown in FIG. 9 is suitable for experimentalpurposes connected with the same requirements for use as indicatedabove. The diffusible, active substance is allowed to drip down fromabove, into cavity 8, at the funnel-shaped, widened part of the tubularsupporting element 3. The substances diffuse into the substrate massthrough a perforated inner part 7 of the supporting element, while aclosed, upper part 9 prevents the substances from leading out onto thesurface of the substrate. In this way, the substances diffuse into thesubstrate along the wall of inner part 7.

The term tubular supporting elements here also refers to supportingelements which are not of circular cylindrical shape and, for example,the supporting elements may lie in the form of an elongated box, asshown at 13 in FIG. 10. Such a shape is suitable if it is required toguide the diffusion in a particular direction. This is achieved byplacing the prepared lining 4 on one of the short walls as indicated inthe figure. The diffusion will then spread along the body 113 of thesupporting element towards the opposite short wall. If the lin ings areplaced along one long side or along both sides as indicated at 14,diffusion equilibrium will be rapidly established. As shown in FIG. 12,the rectangular supporting element in FIG. 10 has neither base norcover, and the supporting element may carry a ferro-magnetic material asshown at ring 15. Further, the supporting element may be provided with aroof or cover, if desired.

FIG. II shows a different embodiment of a supporting element in the formof an elongated, rectangular structure, in which the interior space isdivided by walls 20 into four equal spaces, each of which may have alining containing a different substance. A section through FIG. Illwould have the same appearance as the one through FIG. 10 and shown inFIG. 12.

In the embodiments shown, one of the objects of the invention is to keepthe reaction confined to a certain area. According to the invention, thesupporting elements may, however, also be so designed that only thefirst stage of the diffusion is controlled and guided in a particulardirection, while the final stage is free and unrestricted. An example ofthis is shown in FIGS. 13 and 14. In this case, the supporting element23 is provided on its outer surface with longitudinal concavities 16having small holes 18. These lead into longitudinal passages 19. Thesepassages may either be furnished with active substances duringmanufacture, or the substances to be used or investigated may be addedduring the experiment. This can be achieved by depositing drops of thesubstances into the longitudinal passages 19.

The substances will, in time, diffuse out into the substrate through theholes 18. As the diffusion spreads further outwards, the direction willbe determined by the shape and size of the curved part. It is not untila later stage, when the dispersal has proceeded beyond the confiningborders, that it becomes uncontrolled in all directions.

As explained with reference to FIGS. 4 and 9, the supporting element mayalso be made with a perforated wall lamella confining a chamber intowhich the substances of interest may be introduced. Alternatively, thesupporting element may be without chambers and perforations in thewalls. In such a case, the substances in question may be added in theform of, for example, a paste in the longitudinal concavities I6, orthey may be contained in a lining which, in a suitable manner, is fixedto the outside of the supporting element.

The supporting element shown in FIG. 13 is hollow in the center. Thesupporting element may also be made without this cavity, but if so, itshould be thin enough to ensure that it does not cause cracks to form inthe surrounding substrate during or after its introduction.

In FIG. 15, a star-shaped supporting element is shown wherein aplurality of flat walls 19 intersect along a common line. Thissupporting element causes the substances to be diffused according to afixed pattern. The substances in question are placed or have alreadybeen placed in the angles 17 formed between adjacent walls 19.

The disclosed embodiments merely serve to illustrate the invention andnot to limit it inasmuch as variations and modifications will becomeapparent which will fall within the scope of the invention as defined inthe appended claims.

What I claim is l. A method of carrying out biological and chemicaldiffusion tests in microbiological, serological, immunological, andclinical-chemical work, in which at least one active substance iscontacted with a substrate containing an agent in a container, thereactivity of the agent and active substance being the subject ofinvestigation, said method comprising forming a carrying body with awall which is shaped to confine an area on one side thereof, forcing thecarrying body substantially down to the bottom of the substrate in saidcontainer to confine the substrate in said area, and introducing saidactive substance into the confined substrate by discharging the activesubstance from said body along the depth of penetration of said bodyinto the substrate by diffusion into the substrate whereby the action inthe substrate is restricted to said area which is confined by saidwall,and active substance is not deposited on the upper surface of thesubstrate.

2. A method as claimed in claim 1 wherein said active substance is in alining of solid material on said wall on said one side thereof.

3. A method as claimed in claim 1 wherein the active substance is aliquid, the method further comprising discharging the active substancefrom the body substantially along the entire depth of the wall so thatthe active substance can diffuse into said substrate along the depth ofthe wall.

4. A method as claimed in claim 1 wherein a plurality of bodies is usedand comprising forcing said bodies down into the substrate to the bottomthereof, each body being associated with at least one respective activesubstance.

5. A method as claimed in claim 4 comprising graduating theconcentration of the active substance in plurality of bodies.

6. A method as claimed in claim 4 comprising joining said bodies toforce the same into the substrate simultaneously.

7. A method as claimed in claim 1 wherein said body contains a pluralityof active substances which are introduced into said substrate from saidwall in separate regions thereof.

8. A method as claimed in claim 1 comprising incorporating magnetizablematerial in said carrying body, and forcing the carrying body into thesubstrate by applying an external magnetic force to said body.

9. A method as claimed in claim 1 comprising photometrically analyzingsaid confined area to determine the extent of the reactivity between theagent and active substance.

10. A method as claimed in claim 1 comprising effecting at least onesecondary application of at least one different active substance to thesubstrate subsequent to the time after the first substance and agenthave been brought into contact with one another.

1 l. A method as claimed in claim 1 wherein said area is confined bysaid wall so as to be completely enclosed thereby in a section takenperpendicular to said wall.

12. A method as claimed in claim 1 wherein the carrying body is forcedinto the substrate with its wall substantially perpendicular to thesurface of the substrate.

- 13. A method as claimed in claim 1 wherein said wall is formed with alower edge which extends substantially in a plane.

14. A method as claimed in claim 1 wherein said wall is formed with aheight sufficient to extend above the surface of the substrate when thebody is forced to the bottom thereof.

15. A method as claimed in claim 1 wherein the lower edge of saidcarrying body forms a seal with said container when the body is forcedto the bottom of the substrate.

16. A method as claimed in claim 1 wherein the active substance isdischarged from said wall along the entire depth of penetration of thewall into the substrate.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,791,930 Dated February 12, 197

Rolf Saxholm Inventor (s) It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

In the drawings In figures 10 and 12, the numeral "5" should read 15Signed and Sealed this Fifteenth Day of February 1977 [SEAL] A ttes t:

RUTH C. MASON C. MARSHALL DANN A esting Office Commissioner nfParenIsand Trademarks UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTIONPatent: No. 5,791,930 7 Dated February 12, 197

Inventor s)Rolf SaXhOlm It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

' On the cover sheet item should read:

Norway 1852/69 May 3, 1969 Signed and Scaled this A rtes r:

RUTH C. MASON C. MARSHALL DANN Allesling Officer

2. A method as claimed in claim 1 wherein said active substance is in alining of solid material on said wall on said one side thereof.
 3. Amethod as claimed in claim 1 wherein the active substance is a liquid,the method further comprising discharging the active substance from thebody substantially along the entire depth of the wall sO that the activesubstance can diffuse into said substrate along the depth of the wall.4. A method as claimed in claim 1 wherein a plurality of bodies is usedand comprising forcing said bodies down into the substrate to the bottomthereof, each body being associated with at least one respective activesubstance.
 5. A method as claimed in claim 4 comprising graduating theconcentration of the active substance in plurality of bodies.
 6. Amethod as claimed in claim 4 comprising joining said bodies to force thesame into the substrate simultaneously.
 7. A method as claimed in claim1 wherein said body contains a plurality of active substances which areintroduced into said substrate from said wall in separate regionsthereof.
 8. A method as claimed in claim 1 comprising incorporatingmagnetizable material in said carrying body, and forcing the carryingbody into the substrate by applying an external magnetic force to saidbody.
 9. A method as claimed in claim 1 comprising photometricallyanalyzing said confined area to determine the extent of the reactivitybetween the agent and active substance.
 10. A method as claimed in claim1 comprising effecting at least one secondary application of at leastone different active substance to the substrate subsequent to the timeafter the first substance and agent have been brought into contact withone another.
 11. A method as claimed in claim 1 wherein said area isconfined by said wall so as to be completely enclosed thereby in asection taken perpendicular to said wall.
 12. A method as claimed inclaim 1 wherein the carrying body is forced into the substrate with itswall substantially perpendicular to the surface of the substrate.
 13. Amethod as claimed in claim 1 wherein said wall is formed with a loweredge which extends substantially in a plane.
 14. A method as claimed inclaim 1 wherein said wall is formed with a height sufficient to extendabove the surface of the substrate when the body is forced to the bottomthereof.
 15. A method as claimed in claim 1 wherein the lower edge ofsaid carrying body forms a seal with said container when the body isforced to the bottom of the substrate.
 16. A method as claimed in claim1 wherein the active substance is discharged from said wall along theentire depth of penetration of the wall into the substrate.